The long-term goal of our proposed research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells. Replication of papovaviral DNA in infected cells and in cell-free reactions has proven to be extremely useful as a model system. It has facilitated the identification and characterization of many of the cellular proteins required for replication and has led to a basic understanding of the mechanism of SV40 DNA replication. The specific aims of the research program proposed in this new application are as follows: 1) The phosphorylation state of the SV40 replication protein T antigen governs its ability to initiate viral DNA replication. Work will be performed to test the idea that the phosphorylation state controls cooperative interactions between T antigen hexamers that are required to unwind duplex origin DNA. 2) The role of cell cycle-specific phosphorylation of DNA polymerase alpha-primase and replication protein A (RP-A) in regulating their viral DNA replication activities will be examined. 3) The molecular basis for the species-specificity of polymerase alpha-primase in SV40 and polyoma DNA replication will be investigated. Purified recombinant mouse-human hybrid polymerase alpha-primases will be tested for their ability to replicate viral DNA in vitro. Based on these results, we will attempt to establish cell lines with altered species- specificity for viral DNA replication, and use the hybrid enzymes to study their physical and functional interactions with T antigen and RP-A in vitro. 4) Cis-acting elements that are required for function of a genetic original of DNA replication in mammalian chromosomes will be identified using a competitive PCR approach.